Magnetic resonance imaging (MRI) is an imaging technique that is used in hospitals worldwide. The images are acquired through the use of an MRI scanner and the clinical information is provided through the image contrast, which is based on the magnetic properties in biological tissue. By altering the scanner settings, images with different contrast properties can be obtained. Conventional MRI is a qualitative imaging technique and no absolute measurements are performed. At Center for Medical Imaging and Visualization (CMIV) researchers are developing a new MRI technique named synthetic MRI (SyMRI). SyMRI is based on quantitative measurements of data and absolute values of the magnetic properties of the biological tissue can be obtained. The purpose of this master thesis has been to take the development of SyMRI a step further by developing and implementing a visualization studio for SyMRI imaging of the human brain. The software, SyMRI Brain Studio, is intended to be used in clinical routine. Input from radiologists was used to evaluate the imaging technique and the software. Additionally, the requirements of the radiologists were converted into technical specifications for the imaging technique and SyMRI Brain Studio. Additionally, validation of the potential in terms of replacing conventional MRI with SyMRI Brain Studio was performed. The work resulted in visualization software that provides a solid formation for the future development of SyMRI Brain Studio into a clinical tool that can be used for validation and research purposes. A list of suggestions for the future developments is also presented. Future clinical evaluation, technical improvements and research are required in order to estimate the potential of SyMRI and to introduce the technique as a generally used clinical tool.

Magnetic resonance spectroscopy has been widely used in medical applications, rendering precise evaluation and diagnosis in clinics. As the development of various tools for automatic spectra analysis, providing objective quantification of metabolites, absolute concentrations has been playing an important role in clinical studies and applications as well. In this study, we investigate the reliability and accuracy of absolute concentration quantified by LCModel. Ten healthy subjects were included. We compared the resultant concentrations calculated by internal water scaling and phantom calibration, both of which are provided by LCModel. Partial volume effect was also taken into account to improve the accuracy of absolute concentrations. Automatic segmentation was applied to volume of interest in order to separate gray matter and white matter, which will facilitate the further partial volume correction and thus better accuracy of absolute quantification.

Magnetic resonance spectroscopy is now in widespread use, which with various tools of spectra analysis can provide concentrations of metabolites. The influence of metabolites on human physiology is greatly. Due to the tiny variation of the concentration in various metabolites, the analytic method used in the quantitative determination of the absolute concentrations of metabolites plays an important role in this research area. In this thesis we present an analysis tool for segmentation of white matter, gray matte and cerebrospinal fluid using region growing with spatial space, and provide manual interaction for exception handling in this subject. Then we use this tool to analyze different percentages of white matter and gray matter with the default parameter by LCModel and correct partial volume effect. The results show that the proposed tool can improve significantly the accuracy in absolute quantitative analysis of concentration.

Phosphorus (³¹P) metabolites are emerging liver disease biomarkers. This work aims to develop a quantification protocol for human hepatic ³¹P magnetic resonance spectroscopy (MRS) at 7 tesla (T). It should have high SNR, deliver robust measurements of metabolite concentrations with high reproducibility, and be feasible to use in clinical studies. This will allow detailed characterization of liver metabolism in diseases such as cirrhosis, increasing the utility of ³¹P-MRS as a clinical tool. A 3D chemical shift imaging method using a 16 channel ³¹P array at 7 T is chosen to give high SNR ³¹P spectra from the human liver in vivo, while also providing good spatial localization and spectral resolution. The Oxford Spectroscopy Analysis (OXSA) toolbox, our MATLAB-based processing software package, is introduced and adaptations for analysis of liver spectra are described. Five volunteers were scanned to determine T₁s for the ten visible ³¹P metabolites. Simulations were used to determine design criteria for calibration phantoms at 1.5, 3 and 7 T. I compare three candidate approaches to give "absolute" concentrations in mmol/L wet tissue using a 10 cm loop coil, and then extend these approaches to data acquired using the 16 element receive array. The final protocol was applied to data acquired in ten healthy volunteers and eleven patients with cirrhosis to determine reproducibility and the differences between healthy and diseased livers. This protocol allows distinction between healthy and cirrhotic livers with 90% specificity and sensitivity, using cut-offs in either Î3-adenosine triphosphate or inorganic phosphate concentrations. This ³¹P-MRS absolute quantification protocol is an important first step in fully utilising the increased SNR afforded by the 7 T scanner, offering valuable insight into liver metabolism, and paving the way for other novel ³¹P-MRS methods to be developed in the liver at 7 T.

LCModel has been widely used for MR spectroscopy analysis. LCMgui, which is the built-in user interface of LCModel, based on Linux system, provides the functionality to convert MRS data of various formats to match the format of LCModel raw file, except for GE MRSI data which can be analyzed by LCModel only with GE Sage/IDL software. Hence, the first part of this work was to develop a multi-platform tool with LCModel to support all GE data, including GE MRSI data and phased array data. With this tool, users can analyze MRS data with LCModel on their familiar environment such as Windows, and Linux. The MR spectroscopy experiments with phased array coils provide optimized SNR which lead to more accurate absolute quantification by some sophisticate combination algorithms of phased array coils. Thus, the second part of this work was to propose an algorithm of combining data obtained from phased array coils by doing phase correction and calculation of weighting factor. In addition, the comparison of the accuracy between using quadrature coil and phased array coils with different combination algorithms was investigated in order to demonstrate the efficiency of using phased array coils and the combination program.

The colorectal cancer (CRC) is the third most common malignancy in the world and in Brazil, the fifth most diagnosed and the third cause of cancer deaths. The detection of molecular markers in peripheral blood applies to the early diagnosis of cancer, before and after surgery, reducing the time of identification of CCR and improving the treatment, it is less invasive and reducing costs. This work is quantified by Real Time PCR in absolute and direct, free of DNA fragments found in the serum of patients with colorectal cancer, thereby determining values that characterize the condition of carrier of tumor. For this, a method was developed in which the measurement used as reference samples of the actual fragments ALU115 and ALU247 purified and quantified. We studied 3 major groups: Control, with healthy volunteers; surgery, patients who have already been submitted to surgical removal of colorectal tumor and non-surgery, patients who have not removed the tumor through surgery. Observing the results we note that the groups differ mainly by the values of quantification ALU247. For the control group, the limits were between 91 fentogramas and 1.55 picograms. The non operated group had amounts ranging from 8.02pg to 23.54pg and the group operators, 80fg to 5.95pg. In contrary, the results presented by quantification ALU115 did not set limits defined capable of differentiating at least one of the groups. The control group presented as limits 2pg and 69.45pg and the non operated group, 9.71pg and and 381.56pg, the group operated, 10.69pg and 196.85pg. The non operated group showed a mean significant when compared to the other two groups, but showed minor differences in the quantification ALU247 (14.62 ± 4.73 pg - p <0.05). It could be concluded that direct measurement, using reference concentrations of the fragments themselves ALU115 and ALU247 is a simple methodology, low cost and with reliable results. Moreover, the quantification ALU247 is able to identify the condition of carrier of tumor. / Fundação de Amparo a Pesquisa do Estado de Alagoas / O câncer colorretal (CCR) é a terceira neoplasia maligna mais frequente no mundo, sendo no Brasil a quinta mais diagnosticada e a terceira causa de morte por câncer. A detecção de marcadores moleculares no sangue periférico aplica-se ao diagnóstico precoce de neoplasias, antes e após procedimento cirúrgico, diminuindo assim o tempo de identificação do CCR e melhorando o tratamento, que se torna menos invasivo e diminuindo os custos. O objetivo deste trabalho é quantificar, através da PCR em Tempo Real de forma absoluta e direta, os fragmentos de DNA Livre encontrados no soro de pacientes com câncer colorretal, determinando assim valores que caracterizam a condição de portador de tumor. Para isso, foi desenvolvido um método em que a quantificação utiliza como amostras de referências os próprios fragmentos ALU115 e ALU247 purificados e quantificados. Foram estudados 3 grandes grupos: Controle, com voluntários saudáveis; Operados, pacientes que já se submeteram à cirurgia para retirada de tumor colorretal; e Não-Operados, pacientes que ainda não retiraram o tumor através de cirurgia. Observando os resultados podemos notar que os grupos se diferenciam principalmente através dos valores da quantificação ALU247. Para o grupo Controle, os limites ficaram entre 91 fentogramas e 1,55 picogramas. O grupo Não-Operados apresentou quantidades que vão de 8,02pg a 23,54pg e, o grupo Operados, de 80fg a 5,95 pg. De forma contrária, os resultados apresentados pela quantificação ALU115 não permitiram estabelecer limites definidos capazes de diferenciar pelo menos um dos grupos. O grupo Controle apresentou como limites 2pg e 69,45pg; o grupo Não-Operados, 9,71pg e 381,56pg e; o grupo Operados, 10,69pg e 196,85pg. O grupo Não-Operados apresentou uma média significativa quando comparado aos outros dois grupos, porém apresentou menor heterogeneidade na quantificação ALU247 (14,62pg ± 4,73 - p<0,05). Foi possível concluir que a quantificação direta, utilizando como concentrações de referência os próprios fragmentos ALU115 e ALU247, mostrou-se uma metodologia simples, de baixo custo e com resultados confiáveis. Além disso, a quantificação ALU247 é capaz de identificar a condição de portador de tumor.

En recherche clinique ou environnementale, les biomarqueurs protéiques présentent un intérêt croissant. Bien que les immuno-dosages restent les méthodes de référence pour leur quantification, les récentes avancées en spectrométrie de masse (MS) font de cette technique une alternative crédible à l’ELISA. Ce travail apporte quelques éléments méthodologiques pour repousser certaines limitations de la MS. D’abord, deux dosages ont été proposés. Celui réalisé chez G. fossarum représente le premier exemple de dosage de la Vitellogénine chez un invertébré par LC-MS/MS. L’un des défis de la méthode présentée est de doser spécifiquement une protéine dans un organisme dont le génome est majoritairement inconnu. Le second dosage concerne les peptides contenant une méthionine. Nous avons développé un protocole d’oxydation des méthionines afin de s’affranchir du biais lié à leur oxydation partielle. Cette méthode a ensuite été appliquée à une protéine impliquée dans la maladie d’Alzheimer (l’Apolipoprotéine E4) dans une cohorte de 673 plasmas. Ce dosage est à ce jour l’une des plus grandes études réalisées par LC-MS/MS et montre toute la robustesse de cette approche. Enfin, l’influence de la phase mobile sur la sensibilité des dosages de peptides a été étudiée : d’abord en phase inverse, où le méthanol est une bonne alternative à l’acétonitrile ; ensuite en HILIC, où les difficultés liées à l’étude d’ions multichargés en milieu majoritairement organique ont été abordées. Les problèmes liés à la capacité de charge des colonnes ont également été soulevés. La chromatographie HILIC reste prometteuse pour la quantification de peptides puisqu’un facteur dix en sensibilité peut être apporté. / Both in clinical and environmental research, protein biomarkers are of growing interest. Although immunoassays are the gold standard for their quantification, recent advances in mass spectrometry (MS) make this technique a viable alternative to ELISA. This work provides some methodological elements to eliminate some limitations of the MS approach. First, two assays have been proposed. The first one achieved for G. fossarum represents the first example of quantification of vitellogenin in an invertebrate by LC-MS/MS. One of the challenges of the presented method is to specifically assay a protein in an organism whose genome is largely unknown. The second assay relates to methionine-containing peptides. A protocol was developped for total oxidation of methionines in order to overcome the bias due to their partial oxidation. This method was then applied to a protein involved in Alzheimer's disease (Apolipoprotein E4) in a cohort of 673 plasma samples. This assay is to date one of the largest study carried out by LC-MS/MS and shows all the robustness of this approach. Lastly, the influence of the mobile phase on the sensitivity of peptides assays was studied: first in reversed phase, where methanol is a good alternative to acetonitrile; then in HILIC, where the difficulties associated with the study of multicharged ions in a mainly organic content were discussed. Problems related to the carrying capacity of the columns were also raised.

L'IRM de perfusion et de spectroscopie restent encore peu utilisées en raison de leur mise en oeuvre difficile et de leur manque de quantification. L'objectif de ces travaux a été d'optimiser et de valider des techniques IRM totalement non invasives chez l'Homme en vue d'applications cliniques permettant une exploration sur un large volume cérébral et une quantification absolue des paramètres de perfusion et du métabolisme cérébraux. Concernant la perfusion, 3 séquences de type marquage de spins,PASL PICORE, PASL FAIR et pCASL, ont été comparées en termes de sensibilité et de reproductibilité. pCASL a ensuite été intégrée dans un protocole de recherche sur des patients atteints de sclérose en plaques ou SEP. Quant au métabolisme cérébral, un protocole a été mis en place afin d'accéder à une quantification absolue et pseudo absolue des métabolites par la normalisation du signal de l'eau issue de la CSI par la densité de protons acquise en IRM. Cette technique a été validée en CSI 2D puis transposée en 3D avec la séquence EPSI sur deux orientations différentes : CACP et CACP+15°afin de constituer des valeurs normatives fiables des métabolites principaux sur tout le cerveau. L'élaboration de ces techniques en spectroscopie a abouti à une étude sur des patients souffrant de SEP démontrant la faisabilité de l'utilisation de ces techniques en clinique. Ces travaux démontrent que la quantification absolue en IRM de perfusion et en IRM de spectroscopie peut être obtenue sur un large volume cérébral de manière fiable sur un système IRM disponible en environnement clinique dans un temps d'acquisition acceptable à travers les corrections diverses spécifiques à chaque imagerie. / Conventional MRI's lack of specificity in clinical routine limits our ability to perform correct diagnoses or follow-ups of pathological diseases. Two forms of NMR imaging, perfusion weighed and spectroscopic imaging provide information about two closely related characteristics :cerebral perfusion and metabolism. However, these techniques are not widely used due to the complexity of implementation and a lack of quantification.The general aim was to optimize and validate completely non-invasive NMR techniques for further human clinical applications in the context of exploring large cerebral volumes and determining absolute or pseudo-absolute quantification of cerebral perfusion and metabolism. Concerning perfusion, three arterial spin labeling sequences, PASL PICORE, PASL FAIR and pCASL, were compared in terms of sensitivity and reproducibility. The pCASL sequence was then integrated to a protocol applied to patients suffering from multiple sclerosis. In relation to metabolism, a protocol was applied in order to access absolute and pseudo-absolute metabolite quantification by water SI normalization from MRI proton density. This technique was validated on 2D CSI and then on 3D with EPSI sequence with two orientations, AC-PC and AC-PC+15 in order to generate reliable normative values of metabolites for the whole brain. The use of those spectroscopic techniques on patients suffering from multiple sclerosis allowed demonstrating the feasibility in clinic.This work demonstrates that reliable absolute quantification in perfusion weighted and spectroscopic imaging can be obtained with extensive coverage and with an acquisition time compatible with the reality of clinical exams.

Les toxines botuliques (BoNTs) sont les substances les plus toxiques connues. Elles sont responsables du botulisme, une maladie rare mais le plus souvent mortelle sans prise en charge médicale. Cependant, les applications médicales des BoNTs sont de plus en plus nombreuses du fait de leurs propriétés paralysantes. Leur toxicité par voie inhalée en fait un des 6 principaux agents du risque intentionnel. Les BoNTs, produites par Clostridium botulinum, se répartissent en 7 types sérologiques qui se déclinent en sous-types. Cette biodiversité rend difficile leur identification par les méthodes classiques utilisées pour les toxines protéiques (approches immunologiques). Jusqu’à présent, seule l’analyse génétique permettait de distinguer les différents sous-types entre eux. Dans ce travail a été développée une méthode d’analyse en LC-QqQ-MS/MS en mode MRM pour identifier les différents sous-types de la BoNT/A dans des matrices complexes à partir de peptides communs et spécifiques à ces sous-types. Un traitement d’échantillon par immunocapture sur billes magnétiques couplées à des anticorps anti-peptides a été développé pour isoler la toxine de l’échantillon avant analyse. Des surnageants de culture des sous-types A1 à A3, A5, A7 à A8 ont été utilisés pour valider la méthode. La limite de détection de la méthode est compatible avec les taux de toxine retrouvés habituellement dans les échantillons naturellement contaminés. Cette méthode de spectrométrie de masse a ensuite été utilisée pour quantifier les différents sous-types de la BoNT/A dans une matrice complexe (surnageants de culture de C. botulinum). Une technique de quantification, utilisant un isotope stable de la chaine légère de type A1, ([13C6]K et [13C6]R), a été retenu comme étalon interne. Les différents sous-types de BoNT/A ont été quantifiés dans les surnageants et la quantité de BoNT correspondante à une dose létale minimale de 100% a été déterminée pour chaque sous-type. / Botulinum neurotoxins (BoNTs) are the most poisonous substances known. They are responsible for human botulism, a rare but potentially fatal disease if not quickly treated. However, BoNTs were approved for the treatment of numerous medical applications due to their temporary paralysis effects. BoNTs are among the six agents with the highest risk of potential use as bio-weapons because of their high toxicity in aerosol form. BoNTs, produced by Clostridium botulinum, are divised into seven toxinotypes and each toxinotype contains several subtypes. This biodiversity makes more difficult their identification with classical methods by immunological ways. Until now, only molecular genetical methods could differenciate subtypes among them. The aim of this work was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) in MRM mode to efficiently discrimate the distinct subtypes from specific and common peptides. Immunocapture sample preparation with antipeptides antibodies was used and allowed the isolation of the toxin from the sample. Subtyping was performed with crude supernatants (BoNT/A1 to /A3, /A5, /A7 and /A8) in order to validate the method. Limit of detection (LOD) of the proposed method is in the range of minimal toxin concentration found in naturally contamined samples. In a second part of this work, this mass spectrometry method was used to quantify the neurotoxin in complex matrices (supernatants of Clostridium botulinum cultures). Isotope labeled light chain (13C6]K et [13C6]R) from botulinum A1 neurotoxin was produced and used as internal standart. Subtypes were quantified in supernatants and the quantity of neurotoxin for one minimal lethal dose 100% was determined for each subtype

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